Immunoglobulins play an important role in diagnostic and therapeutic applications. Sera obtained from hyperimmunized mammals has been extensively used to provide immunoglobulins for use in such applications. In certain cases, avian-derived immunoglobulins provide significant advantages over their mammalian counterparts. For example, avian-derived immunoglobulins may provide a higher level of specificity and a reduced amount of undesirable side effects as compared to immunoglobulins derived from mammalian serum.
A useful source of avian IgG immunoglobulins is the yolk of avian eggs. Not only does egg yolk contain high levels of IgG immunoglobulins but it is less labor intensive to collect immunoglobulin-containing eggs from birds than serum from mammals. However, it is necessary to separate the immunoglobulins from other egg yolk constituents such as lipids and lipoproteins to effectively use avian IgG immunoglobulins in assays and therapeutics.
The IgG immunoglobulins in bird egg yolks are hydrophilic and interspersed with non-aqueous components of the yolk. Present methods for separating IgG immunoglobulins from lipids, lipoproteins and other non-aqueous components in egg yolks use multiple treatments with a separating agent. Such methods are time consuming, and often require the use of specialized equipment. For example, in one technique, the aqueous IgG (IgY) immunoglobulins of the egg yolk are separated from the non-aqueous lipids and other components by multiple precipitation extractions using polyethylene glycol (PEG). Polson et al., Immuno. Communications, 2:495-514 (1980). The remaining PEG is removed by precipitation of the IgG immunoglobulin fraction with ammonium sulfate or ethanol at subzero temperatures.
In another method, the lipids and lipoproteins of the egg yolk are precipitated using multiple extractions with dextran sulfate and calcium chloride. Jensenius et al., J. of Immuno. Methods, 46:63-68 (1981). The IgG immunoglobulins are then precipitated with sodium sulfate, and resolubilized to provide a 70-80% yield of total IgG immunoglobulin concentration in the egg yolk. Another method involves multiple extraction steps using organic solvents at -20.degree. C. Bade et al., J. of Immuno. Methods, 72:421-426 (1984). Yet another method utilizes pH adjustment of the egg yolk to separate out the yolk proteins. Jensenius et al., J. of Immuno. Methods, 46:63-68 (1981). The yield of IgG immunoglobulins by this method is only about 50-70% of the total IgG in the egg yolk.
Other methods use separation techniques such as hydrophobic interaction chromatography and gel filtration chromatography. Hassl et al., J. of Immuno. Methods, 110:225-228 (1988). While such methods purport to provide a relatively pure IgG product, purification of the IgG fraction requires expensive separation equipment, and the overall yield of IgG is relatively low compared to other separation procedures.
Accordingly, an object of the invention is to provide a method for separating and purifying a high percentage of the total IgG immunoglobulin from the yolk of an egg without the need for multiple extraction steps, or expensive separation equipment.